title: Extracellular haemoglobin upregulates and binds to tissue factor on macrophages: implications for coagulation and oxidative stress.
abstract: The mechanisms of crosstalk between haemolysis, coagulation and innate immunity are evolutionarily conserved from the invertebrate haemocyanin to the vertebrate haemoglobin (Hb). In vertebrates, extracellular Hb resulting from haemolytic infections binds bacterial lipopolysaccharide (LPS) to unleash the antimicrobial redox activity of Hb. Because bacterial invasion also upregulates tissue factor (TF), the vertebrate coagulation initiator, we asked whether there may be functional interplay between the redox activity of Hb and the procoagulant activity of TF. Using real-time PCR, TF-specific ELISA, flow cytometry and TF activity assay, we found that Hb upregulated the expression of functional TF in macrophages. ELISA, flow cytometry and immunofluorescence microscopy showed binding between Hb and TF, in isolation and in situ. Bioinformatic analysis of Hb and TF protein sequences showed co-evolution across species, suggesting that Hbβ binds TF. Empirically, TF suppressed the LPS-induced activation of Hb redox activity. Furthermore, Hb desensitised TF to the effects of antioxidants like glutathione or serum. This bi-directional regulation between Hb and TF constitutes a novel link between coagulation and innate immunity. In addition, induction of TF by Hb is a potentially central mechanism for haemolysis to trigger coagulation.
authors.forename: Neha
authors.initials: N
authors.lastname: Bahl
authors.forename: Imelda
authors.initials: I
authors.lastname: Winarsih
authors.forename: Lisa
authors.initials: L
authors.lastname: Tucker-Kellogg
authors.affiliation: Prof. Jeak Ling Ding, Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Tel.: +65 6516 2776, Fax: +65 6779 2486, E-mail: dbsdjl@nus.edu.sg.
authors.forename: Jeak Ling
authors.initials: JL
authors.lastname: Ding
mesh_terms: D000975:Antioxidants / Q000378:metabolism; D001777:Blood Coagulation; D002460:Cell Line; D002470:Cell Survival; D003029:Coagulants / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D006454:Hemoglobins / Q000378:metabolism*; D006461:Hemolysis; D006801:Humans; D008070:Lipopolysaccharides / Q000378:metabolism; D049449:Luminescence; D008264:Macrophages / Q000378:metabolism*; D009000:Monocytes / Q000166:cytology; D009154:Mutation; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D000975:Antioxidants; D003029:Coagulants; D006454:Hemoglobins; D008070:Lipopolysaccharides; D017382:Reactive Oxygen Species; D013925:Thromboplastin
keywords: Haemolysis; co-evolution of haemoglobin and tissue factor; lipopolysaccharide; oxidative stress; sepsis